Point-of-care detection of TB and NTM pathogens with Fluorescent Deoxyribozyme Sensors and 3D-printed, battery powered device.

项目摘要 结核病（TB）是由结核分枝杆菌复合群（MTC）引起的一种传染病， 仍然是全球主要的公共卫生问题之一，约有1000万新病例，约有150万 每年死亡。2017年报告了约7万例新病例，巴西仍是22个高负担国家之一 占全球结核病病例的80%以上。非结核分枝杆菌（NTM）是一个新兴的群体 导致TB样肺部感染的相关机会性病原体，特别是在有潜在 合并症如HIV或囊性纤维化。众所周知，NTM感染难以治疗，<50% 尽管使用口服和注射抗生素鸡尾酒长达一年的治疗方案， 毒品艾滋病毒合并感染进一步加剧了这种情况， 使TB和NTM肺部感染的诊断复杂化。估计有350万结核病例未被发现 这主要是由于缺乏负担得起的、有效的结核病/非结核病感染的即时诊断。作为 结果，受感染的病人病情加重，并继续传播这些病原体。频繁 通过常用的涂片显微镜检查方法将NTM感染误诊为结核病， 可能导致更差临床结果的治疗。因此，迫切需要床旁（POC）测试， 为医生提供有效治疗这些使人衰弱的慢性病患者所需的可操作数据， 疾病该项目的目标是利用DNA纳米技术的最新进展， 分子传感器和3D打印，以显着提高结核病和NTM的POC诊断能力。我们 该平台结合了RPA等温扩增的速度和灵敏度，二元扩增的特异性， 脱氧核酶（BiDz）传感器，以及3D打印测定装置的经济性和便携性。我们将优化 多色多重传感器检测，用于特异性检测痰液样本中的TB和NTM病原体。一 使用3D打印构建的廉价便携式电池供电设备，用于检测孵育和荧光检测 技术将受到考验。最后，将在巴西进行该诊断测定的“真实世界”验证， 来自HIV+和HIV-队列的样本。如果成功，这种灵活的技术可以用来设计POC 其他传染病的诊断试验。这一国际合作的另一个成果将是 加强巴西的传染病研究能力和基础设施。

Project Summary Tuberculosis (TB), an infectious disease caused by species of Mycobacterium tuberculosis complex (MTC), remains one of the major public health problems worldwide, with ~10 million new cases and about ~1.5 million deaths each year. With ~70,000 new cases reported in 2017, Brazil remains among the 22 high-burden countries that account for >80% of all TB cases worldwide. Non-tuberculous mycobacteria (NTM) are an emerging group of related opportunistic pathogens that cause TB-like pulmonary infections, particularly in patients with underlying comorbidities such as HIV or cystic fibrosis. NTM infections are notoriously refractory to treatment, with <50% cure rates for some species despite year-long treatment regimens with antibiotic cocktails of oral and injectable drugs. This situation is further exacerbated by HIV co-infection which promotes disease progression and complicates diagnosis of TB and NTM pulmonary infections. An estimated 3.5 million cases of TB go undetected each year, largely due to the lack of affordable, effective point-of-care diagnostics for TB/NTM infection. As a result, infected patients develop more severe disease and continue to transmit these pathogens. The frequent misdiagnosis of NTM infections as TB by the commonly used smear microscopy method delays appropriate treatment which can result in worse clinical outcomes. Thus, point-of-care (POC) tests are urgently needed to provide physicians with actionable data required to effectively treat patients with these debilitating chronic diseases. The goal of this project is to take advantage of recent advancements in DNA nanotechnology, molecular sensors, and 3D-printing to significantly improve POC diagnostics capability for TB and NTM. Our platform combines the speed and sensitivity of RPA isothermal amplification, the specificity of binary deoxyribozyme (BiDz) sensors, and economy and portability of a 3D-printed assay device. We will optimize multi-color multiplex sensor assays for specific detection of TB and NTM pathogens from sputum samples. A cheap, portable battery-powered device for assay incubation and fluorescent detection built using 3D-printing technology will be tested. Finally, “real-world” validation of this diagnostic assay will be conducted in Brazil on samples from HIV+ and HIV- cohorts. If successful, this flexible technology could be exploited to devise POC diagnostic assay for other infectious diseases. An added outcome of this international collaboration will be enhancement of the infectious disease research capacity and infrastructure in Brazil.